Transmitter and Receiver Architectures for Molecular Communications: A Survey on Physical Design with Modulation, Coding, and Detection Techniques

Inspired by nature, molecular communications (MC), i.e., the use of molecules to encode, transmit, and receive information, stands as the most promising communication paradigm to realize the nanonetworks. Even though there has been extensive theoretical research toward nanoscale MC, there are no examples of implemented nanoscale MC networks. The main reason for this lies in the peculiarities of nanoscale physics, challenges in nanoscale fabrication, and highly stochastic nature of the biochemical domain of envisioned nanonetwork applications. This mandates developing novel device architectures and communication methods compatible with MC constraints. To that end, various transmitter and receiver designs for MC have been proposed in the literature together with numerable modulation, coding, and detection techniques. However, these works fall into domains of a very wide spectrum of disciplines, including, but not limited to, information and communication theory, quantum physics, materials science, nanofabrication, physiology, and synthetic biology. Therefore, we believe it is imperative for the progress of the field that an organized exposition of cumulative knowledge on the subject matter can be compiled. Thus, to fill this gap, in this comprehensive survey, we review the existing literature on transmitter and receiver architectures toward realizing MC among nanomaterial-based nanomachines and/or biological entities and provide a complete overview of modulation, coding, and detection techniques employed for MC. Moreover, we identify the most significant shortcomings and challenges in all these research areas and propose potential solutions to overcome some of them.This work was supported in part by the European Research Council (ERC) Projects MINERVA under Grant ERC-2013-CoG #616922 and MINERGRACE under Grant ERC-2017-PoC #780645

ASYMMETRIC DISTRIBUTED LOCK MANAGEMENT IN CLOUD COMPUTING

Cloud computing have become part of our daily lives. They offer a dynamic environment for costumers to store and access their data at any time in any location. The developments of social networks have led to the necessity to build a solution which is easily accesible and available when required. Cloud computing provide a solution that does not depend on the location and can offer a wide range of services, while being free from failure and errors. Although there is an increase in the usage of the cloud storage services, there is still a significant number of aspects such as instant servers failures, network partitioning and natural disasters that require to be carefully addressed. Another important point that is vital for a sustainable cloud is the implementation of an algorithm which will coordinate and maintain concurrent access and keep shared files free from errors. One of the main approaches to overcome these problems is to provide a set of servers which will act as a gateway between clients and storage nodes. In this thesis we propose a new approach which provides an alternative solution to the main problematics related with cloud storages. The approach is based on multiple strategies for eliminating the problem of node failure and network partitioning while providing a complete distributed environment. In our approach, every server acts as a master server for its own requests and can provide service to its clients without interacting with other master servers. The concurrent access is maintained in an asymmetric way through our lock manager algorithm with the least communication among other master servers. According to the state of a specific file, master server can execute any received request without communicating with other master servers and only when additional information is required does further communication occur. In our approach the network partitioning or failure of one or more master servers has no effect on the other part of the cloud. To improve availability, we associate every master server with a failover server which takes up the duty of a master when the master server fails or becomes obsolete. To measure the performance of our approach we have performed various tests and the results are presented in detailed graphs

Improving cell-free protein synthesis for rapid screening applications

Cell-free protein synthesis (CFPS) is a versatile tool for protein research and biomanufacturing. This work goes into great detail explaining the history and biochemical utility of supplements used in various energy mixes. The biological role of each component is discussed in a way that is easily understood by newcomers to the field. This work also takes a unit operations approach to simplifying extract preparation, as well as a novel method for DNA amplification. E. coli cell growth was optimized using a face centered cubic designed experiment that provided an IPTG induction time of 201 min and a harvest time of 255 min. These times correspond to 1 L of growth culture in a 2.5 L shake flask. Experiments were then conducted to determine the optimal number passes through a French press homogenizer (1) as well as the best time and temperature combination for lyophilization (4 hr and 15 Celsius). The resulting extract was more effective than that of commercial kits. This can be used in conjunction with a novel DNA amplification method that modifies a minimal linear template for use in rolling circle amplification. This minimalist template showed identical expression levels to traditional plasmid-based expression using sfGFP. This template was used to successfully express multiple proteins from various classes: sfGFP, mVenus, mCherry, four previously uncharacterized GFP variants, chloramphenicol acetyl transferase, a chitinase catalytic domain, subtilisin, an anti-GFP nanobody, BP100, and CA(1-7)M(2-9). Future directions for the use of CFPS in developing fusion proteins and nanobodies for therapeutics will also be discussed

Discovering probiotic microorganisms: invitro, invivo, genetic and omics approaches

Over the past decades the food industry has been revolutionized toward the production of functional foods due to an increasing awareness of the consumers on the positive role of food in wellbeing and health. By definition probiotic foods must contain live microorganisms in adequate amounts so as to be beneficial for the consumer’s health. There are numerous probiotic foods marketed today and many probiotic strains are commercially available. However, the question that arises is how to determine the real probiotic potential of microorganisms. This is becoming increasingly important, as even a superficial search of the relevant literature reveals that the number of proclaimed probiotics is growing fast. While the vast majority of probiotic microorganisms are food-related or commensal bacteria that are often regarded as safe, probiotics from other sources are increasingly being reported raising possible regulatory and safety issues. Potential probiotics are selected after in vitro or in vivo assays by evaluating simple traits such as resistance to the acidic conditions of the stomach or bile resistance, or by assessing their impact on complicated host functions such as immune development, metabolic function or gut–brain interaction. While final human clinical trials are considered mandatory for communicating health benefits, rather few strains with positive studies have been able to convince legal authorities with these health claims. Consequently, concern has been raised about the validity of the workflows currently used to characterize probiotics. In this review we will present an overview of the most common assays employed in screening for probiotics, highlighting the potential strengths and limitations of these approaches. Furthermore, we will focus on how the advent of omics technologies has reshaped our understanding of the biology of probiotics, allowing the exploration of novel routes for screening and studying such microorganisms

Regulation of the lifestyle switch in Pseudomonas putida

Memoria presentada por Alicia Jiménez Fernández para optar al grado de Doctora en Biotecnología y Tecnología Química por la Universidad Pablo de Olavide de Sevilla.[EN]: In nature most bacteria live as part of structured sessile communities growing on solid surfaces or interphases and embedded in a self-produced extracellular matrix, named biofilms. Pseudomonas putida is a rhizosphere-associated soil bacterium that forms biofilms on multiple biotic and abiotic surfaces. Biofilm formation by P. putida involves cessation of flagellar motility and the production of an extracellular matrix including the high molecular weight adhesins LapA and LapF and several types of extracellular polysaccharides. When nutrients become limitant, P. putida undergoes quick dispersal, during which cells are released from the extracellular matrix and resume a motile lifestyle. Dispersal involves the proteolytic cleavage of LapA triggered by a drop in the intracellular concentration of the second messenger c-di-GMP. In this project we have identified three proteins, BifA, DksA and MvaB, as novel elements involved in the starvation-induced dispersal response. We have shown that the PDE BifA is responsible for decreasing c-di-GMP levels during nutrient starvation and triggering biofilm dispersal. BifA also contributes to the regulation of the steady-state c-di-GMP levels. Positive regulation of bifA expression by the stringent response and the flagellar σ factor FliA links nutrient limitation and the resumption of flagellar motility with c-di-GMP mediated dispersal. We have also studied the role of the c-di-GMP-responsive-σ54-dependent transcriptional factor FleQ as the switch between the planktonic and sessile lifestyles in P. putida. FleQ activates the expression of σ54-dependent flagella and chemotaxis-related promoters and its activity is inhibited by c-di-GMP. At the same time, FleQ exerts dual positive and negative regulation on the transcription of lapA and the cellulose biosynthesis operon in a ¿54-independent manner, and in the case of lapA transcription is stimulated by c-di-GMP. In addition, FleQ and/or c-di-GMP also regulate the synthesis of several putative c-di-GMP-metabolizing enzymes likely contributing to the robustness of the regulatory circuit. Our results indicate that the switch between the planktonic and biofilm lifestyles involves the coordinate regulation of flagellar motility and synthesis and degradation of matrix components. Such regulation involves a plethora of regulatory and signal transduction elements, and is driven by changes in the intracellular levels of c-di-GMP.[ES]: En la naturaleza, la mayoría de las bacterias viven formando parte de biofilms, comunidades sésiles estructuradas embebidas en una matriz extracelular autogenerada que crecen en superficies sólidas o interfases. Pseudomonas putida es una bacteria de suelo asociada a la rizosfera que forma biofilms en superficies bióticas y abióticas. La formación de biofilm en P. putida implica el cese de la movilidad flagelar y la producción de una matriz extracelular que incluye las proteínas de alto peso molecular LapA y LapF y varios tipos de exopolisacáridos. Cuando los nutrientes se agotan, P. putida experimenta un rápido proceso de dispersión en el que las células se liberan de la matriz extracelular y retoman un estilo de vida móvil. La dispersión implica la proteólisis de LapA desencadenada por un descenso en la concentración intracelular del segundo mensajero di-GMPc. En este trabajo hemos identificado tres proteínas, BifA, MvaB y DksA, como nuevos elementos implicados en la respuesta de dispersión inducida por agotamiento de nutrientes. Hemos demostrado que la fosfodiesterasa BifA es responsable de disminuir los niveles de di-GMPc durante la limitación de nutrientes y de desencadenar la dispersión del biofilm. BifA también contribuye a la regulación del estado estacionario de los niveles de di-GMPc. La expresión de bifA está controlada positivamente por la respuesta estricta y el factor σ de flagelo FliA, relacionando así la limitación de nutrientes y la reanudación de la movilidad flagelar con la dispersión mediada por di-GMPc. En este proyecto también hemos estudiado el papel del factor transcripcional dependiente de σ54 y receptor de di-GMPc FleQ como interruptor de la transición entre los estilos de vida planctónico y sésil de P. putida. FleQ activa la expresión de promotores dependientes de σ54 relacionados con la síntesis de flagelo y la quimiotaxis, actividad inhibida por el di-GMPc. Al mismo tiempo, FleQ ejerce una doble regulación positiva y negativa en la transcripción de lapA y del operón para la biosíntesis de celulosa de forma independiente a σ54, y en el caso de lapA la transcripción está estimulada por el di-GMPc. Además, FleQ y/o el di-GMPc también regulan la síntesis de varias enzimas supuestamente relacionadas con el metabolismo del di-GMPc, lo cual probablemente aumenta la robustez del circuito de regulación. Nuestros resultados indican que el cambio entre el estilo de vida planctónico y en biofilm de P. putida implica una regulación coordinada de la movilidad flagelar y la síntesis y degradación de componentes de la matriz. Dicha regulación implica una gran cantidad de elementos de regulación y transducción de señales, y está dirigida por cambios en los niveles intracelulares de di-GMPc.I would like to thank CSIC for the JAE fellowship awarded to me - this kind of funding helps Spanish science grow up and I hope that, in the upcoming years, the number of fellowships increases as well as the public investment in research.Peer Reviewe

Rhythms and Evolution: Effects of Timing on Survival

The evolution of metabolism regulation is an intertwined process, where different strategies are constantly being developed towards a cognitive ability to perceive and respond to an environment. Organisms depend on an orchestration of a complex set of chemical reactions: maintaining homeostasis with a changing environment, while simultaneously sending material and energetic resources to where they are needed. The success of an organism requires efficient metabolic regulation, highlighting the connection between evolution, population dynamics and the underlying biochemistry. In this work, I represent organisms as coupled information-processing networks, that is, gene-regulatory networks receiving signals from the environment and acting on chemical reactions, eventually affecting material flows. I discuss the mechanisms through which metabolism control is improved during evolution and how the nonlinearities of competition influence this solution-searching process. The propagation of the populations through the resulting landscapes generally point to the role of the rhythm of cell division as an essential phenotypic feature driving evolution. Subsequently, as it naturally follows, different representations of organisms as oscillators are constructed to indicate more precisely how the interplay between competition, maturation timing and cell-division synchronisation affects the expected evolutionary outcomes, not always leading to the \"survival of the fastest\"

Genetic and Chemical Intervention of the BfrB:Bfd Interaction Dysregulate Iron Homeostasis in Pseudomonas aeruginosa and Affect its Broader Metabolism

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is accountable for multiple types of infections, including pneumonia, wound, burn, and urinary tract infections. P. aeruginosa is an emerging threat in the hospital environments and preferentially found in comorbid illnesses. Current treatments for the P. aeruginosa infections recommend the use of combination therapy, a β-lactam with an aminoglycoside or a fluoroquinolone. Also, resistance to such procedures is rapidly emerging. Moreover, P. aeruginosa has been given critical priority for anti-infective development in the 2017 World Health Organization (WHO) report on prioritizing pathogens to guide the discovery of new antibiotics. Iron metabolism in bacteria has gained much more attention as a potential target for antibiotic development. Regulation of iron homeostasis is crucial for cells to have enough iron for growth while avoiding iron-induced toxicity. Iron homeostasis involves iron uptake, storage, and mobilization. Two iron storage molecules co-exist in P. aeruginosa, FtnA, and BfrB. Previous in-vitro studies established the importance of the bacterioferritin associated ferredoxin (Bfd) in iron mobilization from BfrB. The X-ray crystallographic structure and biochemical characterization of the BfrB:Bfd complex identified the hot spot residues of the interaction. The residues E81, E85, and L68 in BfrB, and M1, Y2, and L5 in Bfd majorly contribute to the binding energy. Mutating E81 and L68 to alanine completely inhibited iron mobilization from BfrB. The insights gathered from these in-vitro studies were used in this work to interrogate the importance of the BfrB:Bfd interaction in P. aeruginosa cells. In this work, P. aeruginosa, wild type (PAO1), Δbfd, bfrB(E81A/L68A), and ΔbfrB cells were used to study the significances of the BfrB:Bfd interaction in P. aeruginosa cells. A Native-PAGE method was optimized to image iron in BfrB in P. aeruginosa lysates. This advanced technique allowed us to identify BfrB as the main iron storage protein of P. aeruginosa. The same method was used to image iron storage in BfrB and its subsequent mobilization in P. aeruginosa cells (wild type, Δbfd, and bfrB(E81A/L68A)). The intact BfrB:Bfd interaction capable wild type cells showed maximum accumulation of iron during the early stationary phase, and iron mobilization from BfrB during the late stationary phase. In contrast, the Δbfd and bfrB(E81A/L68A) mutants demonstrated irreversible accumulation of iron in BfrB. Due to the irreversible flux of iron into BfrB, the Δbfd and bfrB(E81A/L68A) mutants developed low cytosolic free iron levels and a high total iron to free iron ratio. The consequences of the genetic blockade of the BfrB:Bfd interaction was further investigated by comparing the expression proteomes of the wild type and Δbfd mutant cells. The iron homeostasis dysregulation affected the iron-dependent and independent metabolic processes in the Δbfd mutant cells. Proteins involved in the TCA cycle, amino acid biosynthesis, oxidative stress regulation, and respiratory chain were under-represented in the Δbfd mutant cells. On the other hand, proteins involved in the iron acquisition systems (pyoverdine, pyochelin, Heme iron acquisition), sulfur assimilation, quorum sensing were over-represented in the Δbfd mutant cells. These findings show that iron homeostasis dysregulation can affect the broader metabolism of P. aeruginosa cells. The chemical intervention of the BfrB:Bfd interaction in P.aeruginosa cells was carried out with small molecule inhibitors, analogs of 4-aminoisoindoline-1,3-dione. These analogs acted on their target, BfrB in P. aeruginosa cells. Moreover, the analogs elicited a concentration-dependent growth retardation, and a pyoverdine hyper-production as a result of cytosolic iron deprivation. Analog-treated cells experienced an irreversible accumulation of iron in BfrB and high total iron content in the treated cells. Hence, the developed 4-aminoisoindoline-1,3-dione derivatives were potent to dysregulate iron homeostasis in P. aeruginosa cells. The BfrB:Bfd inhibitors also potentiated the activity of fluoroquinolones in P. aeruginosa (PA01), and in two cystic fibrosis isolates, MR3B and MR60. The growth impairment of the cystic fibrosis and urinary tract clinical isolates of P. aeruginosa, and Acinetobacter baumannii (AB5075) demonstrate the potential widespread application of the developed 4-aminoisoindoline-1,3-dione derivatives in blocking the BfrB:Bfd interaction. These findings strongly support the suitability of inhibiting the BfrB:Bfd interaction as a novel target in anti-infective development

First molecular and biochemical characterization of the extracellular matrix of Saccharomyces cerevisiae

Programa Doutoral em Biologia Molecular e Ambiental (área de especialização em Biotecnologia Molecular)A levedura Saccharomyces cerevisiae, tal como todos os microrganismos, é usualmente considerada como um organismo unicelular. Contudo, os microrganismos formam mais frequentemente comunidades multicelulares macroscópicas que apresentam diferenciação celular, e são coordenadas por um complexo sistema de comunicação, e suportadas por uma matriz extracelular (MEC). A presença deste tipo de suporte das comunidades multicelulares de S. cerevisiae foi descrita no início deste século. Apesar disso, a informação relacionada com a sua composição e organização tridimensional é escassa. Assim, o principal objetivo deste trabalho foi realizar a primeira abordagem sistemática aos principais componentes da MEC de levedura. Para o efeito, foram desenvolvidas metodologias para (1) obter de forma reprodutível uma considerável e homogénea biomassa de leveduras produtora de MEC, e (2) extrair e fracionar a MEC produzida de forma a obter frações analiticamente puras de proteínas e polissacáridos, compatíveis com a aplicação de metodologias analíticas de alto-débito como o GC-MS e o DIGE. A análise detalhada da fração proteica permitiu a identificação de mais de 600 proteínas. A maioria destas tem função e localização intracelulares, e é aqui identificada extracelularmente pela primeira vez, o que pode indicar um moonlighting surpreendentemente elevado. A presença de todas as enzimas associadas à glicólise e à fermentação, assim como ao ciclo do glioxilato, levanta suspeitas sobre a possibilidade de haver metabolismo extracelular. Além disso, um grande número de proteínas associadas à síntese, remodelação e degradação de outras proteínas foi identificado, incluindo elementos da família HSP70 e várias proteases. De realçar a presença das exopeptidases Lap4, Dug1 e Ecm14, e das metaloproteinases Prd1, Ape2 e Zps1, que partilham um domínio funcional zincin com as metaloproteinases da MEC de Eucariotas superiores. A presença adicional de proteínas intervenientes em várias vias de sinalização, como as Bmh1 e Bmh2, e da homing endonuclease Vde, que partilha o domínio Hedgehog/inteína com os morfogenos de Eucariotas superiores, sugere que a MEC de levedura poderá, tal como nesses organismos, mediar sinalização intercelular. As análises cromatográfica e eletroforética da fração glicosídica revelaram claramente a presença de dois polissacáridos. A análise por espectrometria de massa identificou glucose, manose e galactose na composição destes polissacáridos. Foram ainda observados indícios da presença de ácido urónico. A indução de metacromasia sugeriu que os polissacáridos detetados apresentam substituição química. A possibilidade desta corresponder a sulfatação foi testada através de um teste de atividade anticoagulante. Das diversas amostras de MEC de diferentes estirpes de levedura usadas, o duplo mutante gup1Δgup2Δ apresentou, ao contrário da estirpe Wt, razoável atividade anticoagulante indicadora da presença de grupos sulfato. Os efeitos da deleção do gene GUP1 na composição da MEC de levedura proporcionaram uma perspectiva mais detalhada da composição molecular e mecanismos a ela associados. Observaram-se alterações nas frações protéica e glicosídica. A deleção resultou na ausência de várias proteínas, associadas principalmente com o metabolismo de fontes de carbono, defesa e resgate da célula, bem como síntese, modificação e degradação de proteínas, e organização celular. Adicionalmente, a deleção deste gene também teve um grande impacto na composição glicosídica da matriz, levando ao desaparecimento do polissacárido de maior peso molecular detetado na estirpe Wt. Globalmente, os efeitos da deleção do GUP1 na MEC mostram que a estrutura desta é muito dinâmica e que se encontra sob controlo apertado das células que compõem o agregado multicelular. As funções sugeridas para as proteínas ortólogas das Gup1 e Gup2 de levedura, respetivamente Hhatl e Hhat, nas vias de sinalização de Eucariotas superiores esteve na origem da construção de uma bateria de estirpes de levedura recombinantes transformadas com os ortólogos da via Hedgehog de ratinho, mosca e homem, para futura avaliação. Da mesma forma, foram clonados em S. cerevisiae os recetores de mamífero para o ácido hialurónico (AH), CD44 e HMMR. Estes transformantes foram submetidos ao crescimento na presença de AH de diferentes tamanhos moleculares. As estirpes exprimindo ambos os recetores foram igualmente sensíveis à presença de AH de elevado peso molecular, mas foram diferentemente sensíveis à presença de AH de tamanho molecular intermédio. As células expressando o recetor CD44 mostraram-se, tal como em Eucariotas superiores, sensíveis à presença de AH 50 kDa, apresentando uma forte redução da taxa específica de crescimento. Isto indica a expressão funcional dos recetores de AH em levedura e a provável conservação da maquinaria celular de resposta a este componente da MEC dos Eucariotas superiores. Este trabalho é o primeiro a apresentar um estudo detalhado sobre as frações protéica e glicosídica secretadas para a matriz extracelular de S. cerevisiae durante o seu crescimento em comunidades multicelulares, oferecendo a primeira abordagem proteómica e glicómica da sua composição e organização. Globalmente, este trabalho permite prever que a MEC de levedura exerça funções equivalentes às conhecidas da MEC de Eucariotas superiores.The yeast Saccharomyces cerevisiae, as all microbes, is generally regarded as a unicellular organism. However, microorganisms live more frequently in macroscopic multicellular aggregates, presenting cellular differentiation, coordinated by complex communication, and supported by an extracellular matrix (ECM). The presence of this type of structure supporting multicellular life-style of S. cerevisiae was first described early this century. However, the information available on the yeast ECM components and three-dimensional spatial organization is scarce. Hence, this work aimed to provide a first methodical insight into the molecular composition of the yeast ECM major components. A methodology was developed capable of reproducibly obtaining ECMproducing homogenous yeast mats, and extracting and fractionating the yeast ECM into analytical-grade fractions. This was developed in order to be fully compatible with the application of high-throughput analytical techniques, like GC-MS and DIGE. The in-depth analysis of the proteins in the yeast ECM identified more than 600 proteins, most of which being ascribed to intracellular functions and localization, and therefore found extracellularly for the first time. This might indicate unexpectedly extensive moonlighting. The entire sets of enzymes from glycolysis and fermentation, as well as gluconeogenesis through glyoxylate cycle were highly represented, raising considerable reason for doubt as whether extracellular metabolism might exist. Moreover, a large number of proteins associated with protein fate and remodelling were found. These included several proteins from the HSP70 family, and proteases, importantly, the exopeptidases Lap4, Dug1 and Ecm14, and the metalloproteinases Prd1, Ape2 and Zps1, sharing a functional zincin domain with higher Eukaryotes ECM metalloproteinases. The further presence of the broad signalling cross-talkers Bmh1 and Bmh2, as well as the homing endonuclease Vde that shares a Hedgehog/intein domain with the Hh morphogens from higher Eukaryotes, suggest that analogously to the tissues in these organisms, yeast ECM is mediating signalling events. The chromatographic and electrophoretic analysis of the sugar fraction revealed the clear presence of two distinct polysaccharides. Mass spectrometry identified glucose, mannose and galactose in their composition. Evidence was also obtained of the presence of uronic acids. Both polysaccharides showed chemical substitution, as indicated by metachromasia, and the existence of sulphate groups was assessed through an anticoagulant activity test. From several ECM samples from different yeasts strains surveyed, the double mutant gup1Δgup2Δ displayed a relatively high anticoagulant activity, which was not observed in Wt, likely related to the presence of sulphate groups. The effects of the deletion of GUP1 gene in the composition of yeast ECM were also assessed, providing a more in-depth perspective of the ECM components and molecular mechanisms associated. Alterations in both protein and sugar fractions were observed. The deletion of GUP1 led to the absence of several ECM proteins, mainly associated with the carbon metabolism, cell rescue and defence, protein fate and cellular organization. Additionally, the disruption of this gene impacted in the composition of the ECM sugar fraction, through the disappearance of the higher molecular weight polysaccharide that had been detected in the Wt sample. The effects of GUP1 deletion on the ECM show that its structure is very dynamic, and that it is under the tight control of the cells composing the aggregate. S. cerevisiae Gup1 and Gup2 orthologues have suggested regulatory roles in the Hedgehog signalling pathway from higher Eukaryotes, in which organisms these proteins are known as Hhatl and Hhat, respectively. This led to the engineering the yeast mutants defective on either or both GUP1 and GUP2 by expressing these genes orthologues from fly, human and mouse, yielding a collection of transformants for future assessment. Similarly, the mammalian receptors of hyaluronic acid (HA), CD44 and HMMR, were cloned into the yeast S. cerevisiae. The engineered strains were subjected to growth in the presence of different molecular sizes of HA, and were identically and differentially sensitive to, respectively, high and intermediate molecular weight HA. The strain expressing CD44 presented a high growth sensitivity to the presence of 50 kDa HA as in high Eukaryotes. The HA receptors are therefore functional in the yeast cell, and the cellular machinery to respond to HA stimuli appears to be fairly conserved. The present work is the first to present a comprehensive detailed study on the protein and polysaccharide fractions secreted during growth in S. cerevisiae multicellular aggregates. Overall, this work gives a first insight of the multicellular communities of S. cerevisiae proteomics and glycomics, ascertaining yeast ECM with putative roles derived from its components that resemble ECM from higher Eukaryotes